DEUTERIUM AND TRITIUM ISOTOPE EFFECTS ON THE MICROSOMAL N-DEMETHYLATION OF ETHYLMORPHINE

¹ Clinical Pharmacology Section, Veterans Administration Hospital, Minneapolis, and Departments of Medicinal Chemistry and Pharmacology, University of Minnesota.

Ethylmorphine (EM) and trideuteriomethylnorethylmorphine (EM-CD₃) were incubated with rat liver microsomes and an NADPH-generating system in parallel experiments. N-Demethylation was determined by measuring the formaldehyde formed. After 10 min at 37°C, the average deuterium isotope effect (k^H/k^D) was 1.25. Similar small effects were found when incubations were terminated after 1, 2, 4, 8, and 16 min. The k^H/k^D did not vary when the incubations were buffered over the pH range from 5.6 to 8.4. Standard double-reciprocal plots of EM and EM-CD₃ concentration against the rate of formaldehyde formation yielded a V_max^H/V_max^D of 1.26 and identical K_M values of 0.21 mM. Oxygen uptake experiments in the presence of EM and EM-CD₃ demonstrated that deuterium substitution had no effect on this rate. A tritium isotope effect of 2.49 was calculated by incubating N-methyl-tritiated EM with microsomes, removing unreacted substrate from solution, and determining the distribution of tritium between water and formaldehyde. These data, together with that from previous experiments, demonstrate that the small isotope effects found with deuterated and tritiated EM are genuine rate effects and that two steps contribute significantly to the overall rate of N-demethylation. These are the cleavage of the C—H bond and the donation of a second electron to the proposed cytochrome P-450-substrate-superoxide complex.